The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcA1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines. [Cancer Res 2007;67(17):8358-67]

Lectins on antigen presenting cells are potentially involved in the antigen uptake and the cellular recognition and trafficking. Serial analysis of gene expression in monocyte-derived dendritic cells (DCs), monocytes, and macrophages revealed that 7 of the 19 C-type lectin mRNA were present in immature DCs. Two of these, the macrophage mannose receptor and the macrophage lectin specific for galactose/N-acetylgalactosamine (MGL), were found only in immature DCs, as confirmed by reverse transcriptase-PCR and flow cytometric analysis. By subcloning and sequencing the amplified mRNA, we obtained nucleotide sequences encoding seven different human MGL (hMGL) subtypes, which were apparently derived from alternatively spliced mRNA. In addition, the hMGL gene locus on human chromosome 17p13 contains one gene. A single nucleotide polymorphism was identified at a position in exon 3 that corresponds to the cytoplasmic region proximal to the transmembrane domain. Of all the splicing variants, the hMGL variant 6C was expressed at the highest levels on immature DCs from all donors tested. Immature DCs could incorporate ␣-GalNAc-modified soluble acrylamide polymers, and this was significantly inhibited by pretreatment of the cells with an anti-hMGL monoclonal antibody that blocks the lectin-carbohydrate interaction. We propose that hMGL is a marker of imDCs and that it functions as an endocytic receptor for glycosylated antigens. Dendritic cells (DCs)1 play a pivotal role in the immune system by processing and presenting a variety of antigens to T cells (1). The uptake of exogenous antigens is the first step in this process and is therefore a critical event that influences DC function. The uptake of glycoconjugates by DCs is potentially mediated by lectins, which are carbohydrate-binding proteins. There are at least four distinct lectin families in animal cells known as the C-type, S-type, P-type, and I-type lectins (2). Some lectins are known to participate in molecular and cellular trafficking in a manner that is dependent on the lectin type, its molecular architecture, and its subcellular localization. DCs are known to express a variety of lectins, particularly C-type lectins, but as yet their biological roles in DC function are unclear.How glycosylated antigen presentation is regulated and how this affects the subsequent immune responses has not yet been clarified. This is an important issue to investigate as it may improve our understanding of, for example, anti-tumor immunity to MUC1. MUC1 is a glycosylated membrane protein that frequently expresses truncated O-glycans such as the T (Gal␤1-3GalNAc-Thr/Ser) and Tn (GalNAc-Thr/Ser) antigens. MUC1 is an important candidate vaccine antigen as it is an antigen that is often overexpressed in solid tumors, including carcinoma of the breast, lung, pancreas, colon, and ovaries. MUC1-specific cytotoxic T lymphocytes have been isolated from draining lymph nodes of pancreatic and breast cancer patients, ascitic fluids of ovarian cancer patients, and peripheral blood mononuclear cell...

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.

BackgroundDendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs.Methodology/Principal FindingsIn the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII+CD11c+ cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220−CD8αloCD11b+CD11c+MHCIIhi tissue-derived DC. MGL2+DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2+DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC+MGL2+DDCs, but not FITC+MGL2−DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin.Conclusions/SignificanceThese results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2+ DDCs for initiating CHS.

Dendritic cell (DC) subsets in the skin and draining lymph nodes (LNs) are likely to elicit distinct immune response types. In skin and skin-draining LNs, a dermal DC subset expressing macrophage galactose-type C-type lectin 2 (MGL2/CD301b) was found distinct from migratory Langerhans cells (LCs) or CD103+ dermal DCs (dDCs). Lower expression levels of Th1-promoting and/or cross-presentation-related molecules were suggested by the transcriptome analysis and verified by the quantitative real-time PCR analysis in MGL2+ dDCs than in CD103+ dDCs. Transfer of MGL2+ dDCs but not CD103+ dDCs from FITC-sensitized mice induced a Th2-type immune response in vivo in a model of contact hypersensitivity. Targeting MGL2+ dDCs with a rat monoclonal antibody against MGL2 efficiently induced a humoral immune response with Th2-type properties, as determined by the antibody subclass. We propose that the properties of MGL2+ dDCs, are complementary to those of CD103+ dDCs and skew the immune response toward a Th2-type response.